Single-cell analysis is becoming increasingly important for applications in cancer, stem cell development, and cytotoxicity. Localizing individual cells at defined locations is a critical step in the single-cell analysis process. While it is now possible to fabricate well-defined single-cell patterns using arrays of cell-adhesion proteins, the understanding and control of surface properties, pattern geometry, and assay conditions are crucial for optimal single-cell array construction. Here are seven tips for successfully patterning single cells on cell-adhesive protein arrays.

1. Choose an appropriate surface and the best surface chemistry for attaching cell-adhesive proteins. A functional surface (such as epoxy, aldehyde, or Succinimide) chemically couples the adhesive protein to the substrate, which is critical to withstanding subsequent washing steps and potential surface protein delamination.

2. Use the ideal cell-adhesive protein for the cell to be arrayed. Most cell types are capable of binding to certain specific proteins, but not all cells attach to the various extracellular matrix (ECM) proteins with similar efficiencies. As an example, fibroblasts bind well to fibronectin.

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NIH 3T3 fibroblasts bound to patterns of fibronectin on a glass surface.

3. Tailor the size and shape of the cell-adhesive protein pattern to the size and shape of the cell being attached to it. Large cells will have difficulty binding to very small protein patterns, while multiple small cells (and not the desired individual cell) will potentially attach to larger patterns; neither outcome is desirable. Depositing cell-adhesive protein features in pattern shapes that are conducive to cell growth will also contribute to successful single-cell arrays. For example, when arraying neural-like cells, long lines of cell-adhesive protein features are most suitable. When creating cardiac myocyte single-cell arrays, rectangular box patterns of cell-adhesive proteins will enable cells to bind in the rod-like shape they take on in vivo.

4. For the most robust single-cell cultures, work with optimal cell numbers and feature sizes. Using too few cells for attachment to arrayed cell-adhesive proteins will result in insufficient pattern coverage. Large protein features can accommodate lower cell densities (number of cells per unit area) to achieve single-cell array conditions, but too low of a cell density often results in poor coverage of the patterned features. To most efficiently cover protein patterns with predominantly single cells, small features of cell-adhesive proteins and high cell densities should be used.

5. Select the blocking agent that best minimizes nonspecific binding for the specific study being undertaken. As an example, experiments that involve cell patterning followed by motility assessment will work best with a blocking agent such as bovine serum albumin that facilitates specific cell deposition and cellular migration away from patterned areas. Alternatively, in studies where placing the cells and preventing cell motility is important, engineered polymers with up-facing polyethylene glycol groups might be the best blocking agents. In general, hydrophobic blocking agents are not conducive to cell attachment, cell spreading, or cell motility, while hydrophilic blocking agents promote more cell attachment and cell movement over time.

6. Employ appropriate incubation conditions, especially time and media. An experiment’s particular cell-adhesive protein binding dynamics and blocking conditions must be taken into account to determine optimal cell plating time. If cells bind quickly to a specific cell-adhesive protein, incubation time can be short; if cells bind slowly, longer incubation times are necessary. A balance is needed; allow ample time for cells to bind to cell adhesive proteins but not enough time to permit cells to bind in nonspecific locations. Plating media will have a big impact on cell binding speed. Although serum in the media generally contributes to faster attachment of cells to arrayed proteins, serum can coat the nonpatterned surface and lead to high nonspecific binding. In general, serum-free and low-serum plating media generate the highest success rates when plating adherent cells. After cell patterning, for longer term studies, complete media can be used successfully.

7. Use proper washing procedures. After the initial binding of cells to the arrayed cell-adhesive proteins, adequate washing is critical to the creation of desired single-cell patterns. Wash conditions will be dependent on the binding strength between the cells and the patterned adhesive proteins. Use standard manual pipettes (capable of delivering volumes compatible with the size of the substrate) and customary wash reagents; 1 mL volumes of warm PBS typically work well. Employ a moderate wash flow rate. Strong washing (using a vacuum aspirator or flowing wash solution directly over the top of the patterned cells) will likely remove nonspecifically bound cells but may also separate some cells from their cell-adhesive binding proteins. A less-intensive washing technique (i.e., adding wash solution to the culture dish wall) may leave many cells that can subsequently bind nonspecifically. In general, multiple, gentle washings will allow for optimal coverage of protein patterns without separation of cells from patterned protein.

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